Project Summary Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR superfamily. Recent genetic findings show that polymorphisms in the gene encoding the adhesion G protein-coupled receptor (aGPCR) latrophilin 3 (ADGRL3) are strongly associated with an increased risk of attention deficit hyperactivity disorder (ADHD). Remarkably, in fruit flies, zebrafish and mice, disruption of ADGRL3 expression enhanced locomotor activity. In adgrl3 null mice, striatal dopamine levels were shown to be increased, suggesting that the cross-species hyperactive phenotype may be mediated through enhanced dopamine signaling. Thus, ADGRL3 represents a novel target for the development of drug treatments for ADHD and other neuropsychiatric disorders that involve dopamine dysregulation, such as schizophrenia and addiction. However, ADGRL3 remains orphan with respect to the identity of its agonists and signaling properties, and its role in dopamine neurotransmission is poorly understood. Like other aGPCRs, ADGRL3 contains a 7 transmembrane domain and an extracellular N-terminal architecture comprising an array of protein domains suitable for adhesive interactions with protein ligands. In the current proposal we aim to characterize the basic signaling properties of ADGRL3 and how they can be modulated by adhesive protein ligands. For this purpose we will use a comprehensive suite of GPCR signaling assays combined with an innovative magnetic force assay to mimic the mechanical force potentially exerted in binding of trans-synaptic ligands that function to maintain and modulate synapse morphology and organization. In parallel, we will use a combination of genetic strategies together with imaging and behavioral approaches to characterize the role of ADGRL3 in dopamine neurotransmission in vivo. This multidisciplinary approach will also serve as a platform for future studies aimed at testing the functional impact of disease mutations in ADGRL3 and in screening for modulators of ADGRL3 signaling. To achieve these goals we propose the following specific aims: 1) To identify the G protein pathways controlled by ADGRL3 and to establish the role of the N terminus in signaling and in regulating dopamine neurotransmission using (a) in vitro signaling assays and strategic ADGRL3 mutations, (b) an innovative magnetic tweezer assay to evaluate the influence of mechanical force on the activity of ADGRL3 and (c) a Drosophila behavioral model to characterize the localization and function of ADGRL3 in dopaminergic neurons in vivo. 2) To identify the mechanism by which trans-synaptic ligands modulate ADGRL3 signaling and dopamine neurotransmission by determining the effect of teneurin-1 and other ligands on ADGRL3 signaling using the in vitro and in vivo approaches described above.